The focus of the routine human genetic diagnostic laboratory has traditionally been confined to detection of rather large genomic rearrangements. However, the scope of diagnostic services has recently expanded dramatically through the implementation of new powerful molecular diagnostic techniques like Southern hybridization, quantitative PCR, microarrays, and others, although they are either time consuming, restricted to single regions, or need a second method for approval. Concomitantly, the vast availability of genetic diagnostic data and the requirement for improved patient management has drastically increased demand for rapid laboratory diagnosis, forcing many diagnostic labs to develop their own, non-standardized home-brew assays. Thus, there is now an urgent need for highly multiplexed diagnostic analysis systems for genetic aberrations. Unfortunately, current technologies for mutation detection and especially for gene dosis quantification put strict limits on multiplexing capabilities and do not perform cost-effectively. In response to this challenge, Nesher Technologies Inc. (NTI) proposes to develop a cost-effective, solution-based, highly multiplexed, ultrasensitive and -specific, quantitative, rapid, and fully automated analysis system for neurogenetic disorders based on genomic aberrations (with an ultimate capacity of >1,000 aberrations per standard patient sample). NTI has licensed the intellectual property for a revolutionary ultrasensitive biodetection technology with exquisite single well multiplexing potential, which was developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss). It is based on 3-color alternating laser excitation (3c-ALEX) single molecule fluorescence spectroscopy, whereby two (or three) recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two or three colors constitutes a positive target detection event, allowing molecular identification of diffusing molecules in solution and detection of numerous targets freely simultaneously. Over the Phase I funding period we will demonstrate feasibility by simultaneously distinguishing different micro mutations, duplication and deletion of the 1.4-Mb CMT1A locus as well as single nucleotide polymorphisms (SNPs), that can cause two frequent peripheral neuropathies, Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP). Our specific aims are: 1. Separate detection of duplication and deletion of three coding exons of peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 compared to a normal dosage gene (e.g. beta-actin), as well as separate detection of three known SNPs in the CMT1A region, in selected patient samples. 2. Multiplexed detection of the above mentioned aberrations in selected patient samples. 3. Analysis of 250 archived patient samples (including typical and atypical CMT1A duplications and HNPP deletions), and comparison to a home-brew qPCR- and a multiplex ligation dependent probe amplification (MLPA)-based assay routinely used at the quality control reference lab for CMT and HNPP diagnostics. The proposed development of a highly multiplexed, sensitive and -specific, quantitative, low-cost automated test for both gene dosage variation and mutation detection radically pushes the limits of current technologies by allowing simultaneous detection and quantification of multiple genetic aberrations from a single patient sample. By quickly and accurately identifying the disease-causing aberration(s) among the multitude of possibilities according to the patient's presentation, these tests will overcome limitations of current diagnostic methodologies and dramatically improve laboratory diagnosis of inherited neurogenetic disorders. This will greatly facilitate patient management and offer physicians superior guidance for initiation and monitoring of therapies in context with many neurogenetic diseases, e.g. developmental disorders, mental retardation, movement disorders, speech problems etc., associated with aberrations of the human genome. [unreadable] [unreadable] [unreadable]